Targeting Dystroglycanopathies using Pluripotent-derived Myogenic Progenitors

总结 FKRP相关肌营养不良聚糖病的生化标志是α-肌营养不良聚糖的低糖基化 （α-DG），这导致α-DG与细胞外基质蛋白相互作用的破坏，最终导致 到肌肉萎缩FKRP的隐性突变与肌肉的异质性谱相关 从严重的早发性到轻度的晚发性肢带型肌营养不良症（LGMD 2 I）， 先天性肌营养不良症（MDC 1C），包括严重的Walker-Warburg综合征。呼吸 由于膈肌功能丧失而导致的损伤是LGMD 2 I和MDC 1C的显著并发症。没有 目前存在用于肌营养不良聚糖病的经批准的疗法。人们对即将到来的 重编程诱导多能干细胞（iPS）在治疗遗传性疾病中的治疗潜力。的 该项目的前提是，基于干细胞的治疗由人骨骼肌源性祖细胞衍生， 将用正常功能的肌纤维和肌肉干细胞补充患病的肌肉， 其具有在肌营养不良聚糖病中提供长期治疗效果的潜力。我们已经开发 并广泛验证了从多能干细胞产生可移植骨骼肌祖细胞的方法， 细胞通过条件表达Pax 3或Pax 7。这种方法导致高效地生成 治疗性肌源性祖细胞，当移植到营养不良小鼠时，其局部或全身产生 大量的功能性骨骼肌组织，通常与宿主肌肉结合。重要的是， 一部分移植细胞保持单核状态，并显示出骨骼肌干细胞的关键特征， 包括卫星细胞定位，对再损伤的反应，以及对继发性肌肉再生的贡献。 移植试验因此，我们的技术包括重建功能性骨骼肌的细胞疗法， 在患有肌肉萎缩症的宿主中，我们最近发现， PSC衍生的肌源性祖细胞有助于在心肌细胞中显著的肌纤维和卫星细胞再增殖。 免疫缺陷型FKRPP 448 L-NSG小鼠模型。我们有证据表明， 成功地将这些肌源性祖细胞直接递送到FKRP小鼠的膈肌中。另外我们 已经开发了一种通用的FKRP基因校正策略，将其应用于患者特异性WWS和LGMD 2 I 在体外和体内证实了功能性α-DG糖基化的拯救。在本申请中，我们 提出对开发成功的治疗方法至关重要的研究， 营养不良聚糖病，包括理解1）环境对植入的影响， 移植细胞和2）人类基因编辑的WWS的长期功能和分子特征 和未受影响的iPSC衍生的肌源性祖细胞，对于自体和同种异体的未来都很重要 治疗应用，分别。

Summary The biochemical hallmark of FKRP-associated dystroglycanopathies is the hypoglycosylation of α-dystroglycan (α-DG), which leads to disruption in the interaction of α-DG with extracellular matrix proteins, ultimately leading to muscle wasting. Recessive mutations in FKRP are associated with a heterogeneous spectrum of muscle disorders, ranging from severe early-onset to mild late-onset limb-girdle muscular dystrophy (LGMD2I) to several forms of congenital muscular dystrophy (MDC1C), including severe Walker-Warburg Syndrome. Respiratory impairment due to loss of diaphragm function is a prominent complication of both LGMD2I and MDC1C. No approved therapy currently exists for dystroglycanopathies. There has been tremendous excitement for the therapeutic potential of reprogrammed induced pluripotent stem (iPS) cells in treating genetic diseases. The premise of this project is that stem cell-based therapy consisting of human skeletal myogenic progenitors derived from iPSCs will replenish diseased muscle with normal functional muscle fibers as well as muscle stem cells, which have the potential to provide long-term therapeutic effect in dystroglycanopathies. We have developed and extensively validated a method to generate engraftable skeletal myogenic progenitors from pluripotent stem cells through conditional expression of Pax3 or Pax7. This approach results in highly efficient generation of therapeutic myogenic progenitors, which when transplanted into dystrophic mice locally or systemically produce large quantities of functional skeletal muscle tissue that incorporates normally into the host muscle. Importantly, a fraction of transplanted cells remains mononuclear, and displays key features of skeletal muscle stem cells, including satellite cell localization, response to re-injury, and contribution to muscle regeneration in secondary transplantation assays. Therefore, our technology comprises a cell therapy to rebuild functional skeletal muscle, robust to future damage, in hosts with muscular dystrophy. We have recently shown that mouse and human PSC-derived myogenic progenitors contribute to significant myofiber and satellite cell repopulation in the immunodeficient FKRPP448L-NSG mouse model that we generated. Of therapeutic relevance, we have evidence of successful delivery of these myogenic progenitors directly into the diaphragm of FKRP mice. In addition, we have developed a universal gene correction strategy for FKRP, applied this to patient-specific WWS and LGMD2I iPSCs, and demonstrated in vitro and in vivo rescue of functional α-DG glycosylation. In this application, we propose studies that are critical for the development of successful therapeutic approaches for dystroglycanopathies, including understanding 1) the effect of the environment on the engraftment of transplanted cells and 2) the long-term functionality and molecular characteristics of human gene edited WWS and unaffected iPSC-derived myogenic progenitors, important for both autologous and allogeneic future therapeutic applications, respectively.